(T1430-06-31) Automated Supporting Liquid Extraction (SLE) Workflow to Extract Zolmitriptan from Plasma Samples for Bioanalysis

Purpose: Typical bioanalysis workflows for analyzing small molecules use organic solvents to extract the analyte(s) and precipitate proteins. Sometimes, additional cleanups, such as liquid-liquid extraction or solid phase extraction, may be required to remove more matrix and enrich analytes to improve sensitivity, accuracy, and precision. However, developing a bioanalysis method can be challenging, as some operating procedures can be difficult to speed up and scale up. Therefore, there is a need for standardized and efficient automated sample preparation methods to maintain high sample cleanliness and data quality. Supported Liquid Extraction (SLE) utilizes modified diatomaceous earth to facilitate the partitioning of compounds between immiscible organic and aqueous layers. The biological sample, pretreated with an aqueous buffer, is loaded onto the SLE cartridge (or plate), spreading over a thin surface layer. When the organic solvent is passed through, the analytes partitioned into the organic layer are collected, while the hydrophilic matrix captured in the sorbent is removed. SLE is compatible with laboratory automation and only requires sample pre-treatment, load, wait, and elute steps, providing a simpler and faster solution to enrich analytes.  The purpose of this presentation is to demonstrate the effectiveness of the streamlined and automated SLE sample preparation workflow before LC-MS/MS for bioanalysis.

Methods: To assess the feasibility of using the SLE workflow in bioanalysis, Zolmitriptan, a selective serotonin receptor agonist, was spiked into plasma (Human K₂EDTA) at different concentrations. Briefly, plasma samples were thawed at room temperature and aliquoted into a 96-well sample plate. The sample plate was placed in the Extrahera Automated Sample Prep station to execute the following steps: a) add 0.5 M NH₄OH to each sample, b) thoroughly mix the sample with the pretreatment solvent 3 times; c) transfer pretreated mixtures and load onto the SLE plate; d) apply positive pressure to allow liquid to adsorb into the SLE sorbent; e) wait for 5 minutes; and f) add methyl tert-butyl ether (MTBE) to elute the analyte. The elution fraction (in MTBE) was dried and then reconstituted using acetonitrile: water (5:95, v/v) with 0.1% formic acid for LC-MS/MS analysis.

Results: This automated SLE workflow offered >80% analyte extraction recovery, reduced matrix effects (0.99-1.02), excellent precision (RSD < 6%), and accuracy (with error < ± 6%) within and between multiple batches of SLE. During the method development, we pretreated the plasma sample with 0.5 M NH₃OH to keep Zolmitriptan (pKa 9.64 and Log P 2.2) non-ionized for better solubility in an organic solvent. MTBE and ethyl acetate (EA) were assessed as elution solvents, showing comparable performance. MTBE is better in minimizing the matrix effect, while ethyl acetate is better in recovery. Plasma spiked with different concentrations of Zolmitriptan (from 1-100 ng/mL) were used to establish the calibration curve. We compared the calibration curves run on 3 plates of different batches on different days and achieved excellent reproducibility and linearity (R >0.998). Three levels of QC samples with spiked analyte concentrations (ng/ml) at 8, 60, and 85 were analyzed to evaluate the accuracy and precision within and between the SLE plates of different batches. The results demonstrate excellent precision (3.3-6.0 %) and accuracy (101.3-105.8 %) within and between the plates, even without using internal standards. The phospholipid profile results indicated that the SLE method effectively removed >99% of the phospholipids co-extracted by the protein precipitation method. The SLE process was automatically handled by the Extrahera, an automated sample preparation workstation. The total experiment time to process a 96-well SLE plate took 102 min, while the time a chemist spent on the bench for the experiment was reduced to 60 min.

Conclusion: The automated SLE workflow not only demonstrated excellent performance in matrix effect, recovery, linearity, accuracy, and precision for bioanalysis but also significantly reduced the time a chemist spends on the bench. This reduction in manual labor can greatly improve the efficiency of bioanalytical laboratories.

References: 1. Vishwanathan, Karthick, Michael G. Bartlett, and James T. Stewart. “Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry.” Rapid Communications in Mass Spectrometry 14.3 (2000): 168-172.
2. Little JL, Wempe MF, Buchanan CM. “Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma.” Journal of Chromatography B. 2006 Apr 3;833(2):219-30.